Abstract Background Chimeric antigen receptor (CAR)-T cell therapy remains largely ineffective in glioblastoma (GB), where a highly immunosuppressive microenvironment and tumor heterogeneity impair therapeutic durability. Methods Using a human neocortical brain slice model that preserves the complex GB microenvironment, we profiled interactions between natural killer group 2D ( NKG2D ) CAR-T cells and tumor ecosystems via PIC-seq, spatial transcriptomics, and gene regulatory network reconstruction. Results CAR-T cells showed an early but unsustained tumor-suppressive effect in the slice model. Single-cell profiling revealed that CAR CD8 T cells adopt an effector-skewed activation state accompanied by coordinated upregulation of checkpoint receptors and an exhaustion-associated transcription factor program. These transcriptional changes were linked to ligand-receptor signaling interactions between CAR-T cells and myeloid populations. Tumor-associated macrophages displayed enhanced phagocytic programs and spatially co-localized with mesenchymal-like GB cells within hypoxic regions. Gene regulatory network analysis identified MAF and BACH2 as candidate regulators of CD8 T cell state, with MAF enriched in CAR CD8 T cells exhibiting exhaustion-like features, and BACH2 enriched in Mock CD8 T cells consistent with less differentiated programs. In silico perturbation analyses further suggested a reciprocal effect of MAF and BACH2 on CD8 T cell transcriptional trajectories. Conclusions These data map the microenvironmental and transcriptional changes associated with rapid CAR-T cell dysfunction in GB and identify candidate pathways and regulators that may be leveraged to engineer more durable cellular therapies.
Zhang et al. (Fri,) studied this question.